College of Postgraduate, Hebei North University, Zhangjiakou, Hebei, China.
Department of Geriatric Cardiology, Hebei General Hospital, Shijiazhuang, Hebei, China.
Biosci Rep. 2023 Sep 27;43(9). doi: 10.1042/BSR20230014.
MicroRNA-21 (miR-21) plays an anti-apoptotic role following ischemia-reperfusion (I/R) injury (IRI) in vivo; however, its underlying mechanism remains unclear. The present study explored the effects of miR-21 and homeodomain interacting protein kinase 3 (HIPK3) on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in vitro. To this end, the rat cardiomyocyte H9C2 cell line was exposed to H/R and the roles of miR-21 and HIPK3 in regulating cell viability and apoptosis were evaluated by cell counting kit-8 assay, terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling, and flow cytometry. Immunofluorescence and Western blotting were performed to detect the expression/phosphorylation of apoptosis-related proteins. miR-21 expression was measured with quantitative real-time polymerase chain reaction. The putative interaction between miR-21 and HIPK3 was evaluated using the luciferase reporter assay. Our results showed that (i) miR-21 overexpression or HIPK3 down-regulation significantly attenuated H9C2 cells apoptosis after H/R, (ii) suppression of miR-21 expression promoted apoptosis, (iii) miR-21 overexpression inhibited HIPK3 expression, (iv) HIPK3 was the direct and main target of miR-21, (v) miR-21/HIPK3 formed part of a reciprocal, negative feedback loop, and (vi) HIPK3 down-regulation decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD, which subsequently inhibited the expression of BAX and cleaved caspase-3 and increased the expression of BCL2. Our study indicates that miR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression, which could eventually have important clinical implications for patients with acute myocardial infarction.
微小 RNA-21（miR-21）在体内缺血再灌注（IRI）损伤后发挥抗细胞凋亡作用；然而，其潜在机制尚不清楚。本研究探讨了 miR-21 和同源结构域相互作用蛋白激酶 3（HIPK3）对体外缺氧/复氧（H/R）诱导的心肌细胞凋亡的影响。为此，将大鼠心肌细胞 H9C2 细胞系暴露于 H/R 中，并通过细胞计数试剂盒-8 测定、末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记和流式细胞术评估 miR-21 和 HIPK3 在调节细胞活力和凋亡中的作用。免疫荧光和 Western blot 用于检测凋亡相关蛋白的表达/磷酸化。使用实时定量聚合酶链反应测量 miR-21 表达。通过荧光素酶报告基因测定评估 miR-21 和 HIPK3 之间的假定相互作用。我们的结果表明：（i）miR-21 过表达或 HIPK3 下调可显著减轻 H9C2 细胞 H/R 后细胞凋亡；（ii）抑制 miR-21 表达促进凋亡；（iii）miR-21 过表达抑制 HIPK3 表达；（iv）HIPK3 是 miR-21 的直接和主要靶标；（v）miR-21/HIPK3 形成了一个相互的负反馈循环的一部分；（vi）HIPK3 下调通过抑制 FADD 的磷酸化减少 FAS 介导的凋亡，从而抑制 BAX 和裂解 caspase-3 的表达，增加 BCL2 的表达。我们的研究表明，miR-21 通过调节 HIPK3 表达减轻 FAS 介导的心肌细胞凋亡，这对急性心肌梗死患者具有重要的临床意义。
